2023 Volume 48 Issue 2 Pages 57-64
In non-clinical animal studies for drug discovery, histopathological evaluation is the most powerful tool to assess testicular toxicity. However, histological analysis is extremely invasive; many experimental animals are needed to evaluate changes in the pathology and anatomy of the testes over time. As an alternative, small animal magnetic resonance imaging (MRI) offers a non-invasive methodology to examine testicular toxicity without radiation. The present study demonstrated the suitability of a new, ready-to-use compact MRI platform using a high-field permanent magnet to assist with the evaluation of testicular toxicity. To validate the utility of the MRI platform, male mice were treated with busulfan (40 mg/kg, intraperitoneal injection). Twenty-eight days after treatment, both testes in busulfan-treated and control mice (n = 6/group) were non-invasively scanned in situ by MRI at 1 tesla. On a T1-weighted 3D gradient-echo MRI sequences (voxel size: 0.23 × 0.23 × 0.50 mm), the total testicular volume in busulfan-treated mice was significantly smaller than in controls. On T1-weighted images, the signal intensity of the testes was significantly higher in busulfan-treated mice than in controls. The mice were sacrificed, and the testes were isolated for histopathological analysis. The weight of the testes in busulfan-treated mice significantly decreased, similar to the results of the non-invasive analysis. Additionally, periodic acid-Schiff stain–positive effusions were observed in the interstitium of the busulfan-treated mouse testes, potentially explaining T1 shortening due to a high concentration of glycoproteinaceous content. The present data demonstrated a rapid evaluation of testicular toxicity in vivo by compact MRI.
In the drug discovery process, a pipeline compound that can affect the size and weight of the testes points to a sign of serious testicular toxicity. Therefore, testicular toxicity must be adequately evaluated before the first clinical trial on humans. In testing male reproductive toxicity, using experimental animals is preferable to detect similar changes occurring in humans exposed to the same chemicals. Among all adverse findings, histopathology and the weight of a male reproductive organ are the best non-clinical endpoints to detect reproductive toxicity (Scialli et al., 2018; Sotos and Tokar, 2012). However, it is difficult to determine testicular size from the body surface. Furthermore, these data can only be collected by euthanizing a large number of animals, but reducing the number of animals used in toxicological research is required from the perspective of animal welfare. In addition, histological findings are generally not available in humans. To address these concerns, it is important to develop a non-invasive method to evaluate testicular toxicity in vivo.
Owing to its lack of ionizing radiation and high-contrast resolution, magnetic resonance imaging (MRI) with a high-frequency magnetic field is a powerful, non-invasive tool to detect and monitor the status of lesions with high sensitivity (Pichler et al., 2010). MRI can be applied to small animals in non-clinical toxicological studies. This approach enabled us to evaluate time-course changes in pathological and anatomical findings and their reversibility in the same animals. It can also detect the presence and location of induced lesions and/or deposition of abnormal proteins, assisting with the selection of sections for subsequent histopathological examination. In fact, MRI has been used to detect amyloid plaques by using amyloid-beta precursor protein transgenic mice (Zhang et al., 2004); this demonstrates the usefulness of MRI as a complementary tool for conventional histopathology. Thus, MRI widens the range of potential non-invasive imaging modalities, expanding the scope of non-clinical studies. However, the widespread use of MRI systems in non-clinical studies are hindered by obstacles, such as high costs of purchase and maintenance, significant siting and installation requirements, and complicated operations.
With progress in design technology, a novel compact MRI system with a high-field permanent magnet (~1.0 tesla) has been developed to reduce the cost and complexity of superconducting magnets for MRI systems (Taketa et al., 2015; Pittala et al., 2018; Daneshgaran et al., 2019). The new system is portable and self-shielded; it can be placed in most institutions. Cryogens, plumbing, chemicals, and supplemental power suppliers or coolers are not required. Compared with conventional MRI systems (Natt et al., 2002), the new system has been utilized in several non-clinical studies, including those on hepato-, nephro-, and neurotoxicities (Taketa et al., 2015; Tempel-Brami et al., 2015). However, no evidence has been found on the evaluation of testicular toxicity in vivo by MRI.
The aim of the present study was to examine the usefulness of a ready-to-use, novel compact MRI platform in evaluating testicular toxicity. We used an infertility mouse model commonly used for busulfan administration (Simmons et al., 1975; Nakata et al., 2020; Gutierrez et al., 2016; Wang et al., 2010). Changes in the T1-weighted images of the testes were examined using a compact MRI system and compared with testicular histology results.
In total, 12 male C57BL/6J mice (CLEA Japan, Tokyo, Japan) aged 5 weeks were purchased. In CLEA Japan, all mice had free access to water and standard animal food, and were exposed to a 12-hr light/dark cycle, a temperature of 23 ± 3°C, and a humidity-controlled environment (40–60%). Afterwards, the mice were shipped from CLEA Japan, and maintained under our laboratory conditions. The mice were housed in individually ventilated cage systems (Lab Product Inc., Seaford, DE, USA) at 23 ± 2°C, under a 12-hr light/dark cycle (lights on from 8:00 to 20:00), and with food and water provided ad libitum. Body weight was measured once a week (three mice per cage group). We followed the guidelines for animal care and use established by the Ethical Committee for Animal Experiments of the National Institutes of Health Sciences. The animal facility was approved by the Health Science Center for Accreditation of Laboratory Animal Care, Japan. All experimental protocols in the study were reviewed and approved by the Committee for Proper Experimental Animal Use and Welfare, a peer-review panel established at the NIHS (experimental approval no. 816).
Experimental designAfter 1 week of habituation, the mice, aged 6 weeks, were randomly divided into two groups as follows: the vehicle control (n = 6) and busulfan-treated groups (n = 6). To generate the mouse model of infertility, a single intraperitoneal (i.p.) dose of busulfan exceeding 30–40 mg/kg has commonly been selected (Nakata et al., 2020; Gutierrez et al., 2016; Wang et al., 2010). However, this high i.p. dose of busulfan increases the mortality rate of mice (Wang et al., 2010). In the present study, a two-i.p. dose schedule was designed to avoid the increase in the mortality rate of mice. The vehicle control group received two i.p. injections of a vehicle containing 10% dimethyl sulfoxide (DMSO; 031-24051; CultureSure®; FUJIFILM Wako Pure Chemical Industries, Ltd., Osaka, Japan) in saline (Otsuka Pharmaceutical Co., Tokyo, Japan) with a 3-hr interval between injections. Busulfan (B-2635; Sigma–Aldrich, St. Louis, MO, USA) was dissolved in DMSO at a concentration of 10 mg/mL and was gradually added to nine times the volume of saline (final concentration: 1 mg/mL). As previously described, the busulfan-treated group received two i.p. injections (20 mL/kg per injection) of busulfan at a total dose of 40 mg/kg body weight (Xie et al., 2020). After 28 days (sexual maturation), the mice were anesthetized with isoflurane for MRI analysis. After MRI data were obtained, the reproductive organs of male mice were collected under isoflurane anesthesia. All efforts were made to minimize animal suffering. Each mouse was weighed; blood was collected from the inferior vena cava, and the testes were removed. The dissected tissues were rapidly fixed for histopathological analysis.
In vivo imaging and analysis using a compact MRIBoth busulfan-treated and control mice (n = 6/group) were anesthetized with isoflurane, and the testes were scanned using a compact MRI system (M7 Permanent Magnet System; 1.05 tesla; Aspect Imaging, Shoham, Israel) equipped with an application-specific mouse body radiofrequency coil. For in vivo imaging, the mice were maintained in an anesthetized state with 1.5% isoflurane in O2 and placed on a specially designed heated bed, where the respiratory rate was monitored to determine the anesthetic level (PC_SAM; SA Instruments, NY, USA).
High-resolution datasets were obtained using a T1-weighted 3D gradient-echo MRI sequence (echo time/repetition time, 2.0 msec/20.0 msec; field of view, 27.04 × 27.04 mm; matrix, 117 × 117; voxel size, 0.23 × 0.23 × 0.50 mm; scan time, 119 sec). For quantitative evaluation, the signal-to-noise ratios of selected testis structures were determined. This was defined as the MRI signal intensity divided by the standard deviation of the noise. Testicular volume estimates were obtained by drawing 3D regions of interest in a sophisticated image processing and analysis software package (VivoQuantTM 2020; Invicro, Boston, MA, USA) that is fully integrated into the M7 imaging system.
Testicular histologyFor histopathological analysis, the testes were removed and immersed in Bouin’s fixative (023-17361; FUJIFILM Wako Pure Chemical Industries, Ltd.) for 48 hr. After fixation, the testes were processed in graded alcohol, cleared in xylene, and embedded in paraffin. Sections (4-μm thick) were prepared, deparaffinized, and stained with periodic acid–Schiff–hematoxylin (PAS-H) to distinguish germ cell types and seminiferous tubule stages. The slides were mounted using Entellan® (Nacalai Tesque Inc., Kyoto, Japan) and air-dried prior to microscopic examination. A total of 100 seminiferous tubule sections were observed in two tissue sections, which were obtained from two discrete portions of a testis from a single mouse. We evaluated two tissue sections per mouse (n = 6 per group) by using a light microscope (BX50; Olympus Co., Tokyo, Japan) with the cellSens imaging software. The percentage of germ cell depletion in the seminiferous tubules was assessed.
Statistical analysisData are presented as mean ± standard error of the mean (SEM). Welch’s t-test was used to detect significant differences between the control and busulfan-treated groups using GraphPad Prism software (version 9.4.1) (GraphPad Software, Inc., San Diego, CA, USA). Statistical significance was set at P < 0.05.
Busulfan treatment decreased body weights (control: 25.6 ± 0.3 g, busulfan-treated: 23.6 ± 0.3 g, P < 0.01, Table 1). Fig 1A and B shows the gross appearance of the testes in both control and busulfan-treated mice. The absolute weights of both left and right testes in busulfan-treated mice were significantly lower than those in control mice (Fig. 1E, F, Table. 1).
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Photomicrographs and T1-weighted 3D gradient-echo MR images of the testes. Photomicrographs of the gross appearances of (A) control and (B) busulfan-treated mouse testes. Coronal, maximum-intensity projection images of (C) control and (D) busulfan-treated mouse testes. High-resolution datasets were obtained with the following parameters: echo time/repetition time, 2.0 msec/20.0 msec; field of view, 27.04 × 27.04 mm; matrix, 117 × 117; voxel size, 0.23 × 0.23 × 0.50 mm; scan time, 119 sec. (E, F) Busulfan significantly decreased the weights of the left and right testes. (G, H) Busulfan significantly decreased the total volume of the left and right testes. (I) A correlation between weight of the testes (mg) and the testes volume (mm3) was found. Data are expressed as mean ± SEM (n = 6 each). *P < 0.05, **P < 0.01, ***P < 0.001 vs. control (Welch’s t-test in GraphPad Prism (version 9.4.1)).
Figure 1C and D shows the maximum-intensity projection of T1-weighted gradient-echo images of control and busulfan-treated mice, respectively. Measurements of the images showed a left testicular volume of 61.3 ± 3.2 mm3 (mean ± SEM) in the control mice and 19.8 ± 1.0 mm3 (mean ± SEM) in the busulfan-treated mice (Fig. 1G, Table 1). Measurements of the images showed a right testicular volume of 62.6 ± 2.1 mm3 (mean ± SEM) in the control mice and 19.4 ± 1.0 mm3 (mean ± SEM) in the busulfan-treated mice (Fig. 1H, Table 1). A correlation between the weight of the testes (mg) and the testes volume (mm3) was also found (Fig. 1I). Fig. 2A–D shows the T1-weighted images of mouse testes from which the signal was measured. The signals on T1-weighted images in both left (Fig. 2E) and right (Fig. 2F) testes were significantly higher in busulfan-treated mice than in control mice (Fig. 2E).
T1-weighted 2D gradient-echo images of both control and busulfan-treated mice. T1-weighted 2D gradient-echo images showing the regions of interest in the testes of (A) control mice, indicated by the yellow oval, (B–D) and busulfan-treated mice, indicated by the red ovals. (E, F) Compared to that of control mice, the signal from the left and right testes on T1-weighted images was significantly increased in busulfan-treated mice. Data are expressed as mean ± SEM (n = 6 each). *P < 0.05, **P < 0.01, ***P < 0.001 vs. control (Welch’s t-test in GraphPad Prism (version 9.4.1)).
The histopathological analysis of the seminiferous tubules showed that busulfan-treated mice accounted for approximately half of seminiferous tubules with spermatogenic cell loss; in contrast, this was not observed in control mice (Fig. 3A, B). In busulfan-treated mice, testicular toxicity was characterized by the vacuolization of Sertoli cells and degeneration of spermatocytes and spermatids (Fig. 3B). PAS-positive effusions were also observed in the interstitium of the seminiferous tubules of these mice (Fig. 3B).
Photomicrographs of mouse testes cross-sections. The tissues were stained with periodic acid–Schiff–hematoxylin (PAS-H) and analyzed at 200 × magnification (scale bar = 250 μm). (A) Photomicrographs of cross-sections from control mice. (B) Photomicrographs of cross-sections from busulfan-treated mice. The samples were collected 28 days post-treatment. Most busulfan-treated mice showed depletion of spermatogenic cells and severe disruption of seminiferous tubular structures in the testes; however, approximately 40% of these structures contained only somatic cells. PAS-positive effusions were observed in the interstitium of busulfan-treated mouse testes (arrows in red, n = 6 each). *shows spermatogenic cell loss in the seminiferous tubules.
Although the histopathology and weight of the testes are the best non-clinical endpoints to detect testicular toxicity in experimental animals, these analyses can only be performed after euthanizing a large number of animals. In the view of the 3Rs, the development of a non-invasive testicular toxicity evaluation method is needed to obtain time-dependent toxicity information from the same animal, which will contribute to a reduction in the number of animals used. However, testicular toxicity testing has been a challenge due to the lack of simple and robust screening methods; thus, to evaluate non-clinical toxicity in vivo, the number of animals to be used cannot be reduced. The present study demonstrated the significance of developing a non-invasive method for testicular toxicity evaluation using an infertility mouse model; this model made more feasible predictions even with decreased time and resources.
The present study used well-established, busulfan-treated mice to produce a male infertility model. Busulfan, an alkylating agent, can inhibit cell division by binding to one of the DNA strands, making spermatogenic cells with high cell division rates susceptible to busulfan treatment. A single i.p. injection of busulfan eliminates almost all endogenous spermatogenic cells in mice; this has become the most commonly technique to induce infertility (Nakata et al., 2020; Gutierrez et al., 2016; Wang et al., 2010). To sterilize the mice, a single-dose i.p. injection of busulfan should exceed 30 mg/kg (Nakata et al., 2020; Gutierrez et al., 2016; Wang et al., 2010); however, this notably increases the mortality rate of mice (Wang et al., 2010). Previous studies used 50% DMSO in saline or water as a vehicle control to dissolve busulfan (Nakata et al., 2020; Gutierrez et al., 2016; Wang et al., 2010); however, this cannot be applied to toxicity studies. In the present study, we established a method to dissolve busulfan in 10% DMSO and 90% saline solution. As previously described, to deplete spermatogenic cells without lethality, we administered two i.p. injections of busulfan at 3-hr intervals (1 mg/mL busulfan × 20 mL/kg per injection) and a total dose of 40 mg/kg body weight (Xie et al., 2020); this did not have a lethal effect on a total of 36 mice, even in the preliminary test (data not shown).
Histopathological analysis is the gold standard for evaluating testicular toxicity (Takahashi and Matsui, 1993). In non-clinical studies, changes in testes weight can be a sensitive indicator of moderate damage to the testes (Lanning et al., 2002). However, this approach requires many animals for time-course toxicity testing, which includes observation of recovery from anatomic lesions over time; this necessitates non-invasive methods of measurement. MRI is often used as a non-invasive alternative to evaluate testicular architecture and pathology. A novel, high-performance compact MRI with a high-field permanent magnet has been utilized in several non-clinical studies, encompassing a wide range of applications in hepato-, nephro-, and neurotoxicity studies, among others (Taketa et al., 2015; Tempel-Brami et al., 2015). However, no reports have been made about non-invasive 3D images of the testes from in vivo MRI analysis. To the best of our knowledge, this is the first study to demonstrate the utility of MRI analysis in evaluating testicular toxicity in a mouse model of busulfan-induced spermatogenic disorder; busulfan causes extensive degeneration of seminiferous tubules (de Rooij and Kramer, 1970; Bucci and Meistrich, 1987).
In the present study, we obtained 3D images in vivo by using a T1-weighted 3D gradient-echo MRI sequence. This gapless sequence has several advantages over the standard multi-slice imaging procedure. Gapless image acquisitions are most profitable in grasping small organs, such as mouse testes, because a slice gap is inevitable in the standard multi-slice imaging procedure. Custom image analysis scripts were created for the automated segmentation of the testes in situ. The segments were serially measured, and the total testicular volume was calculated by reconstructing these areas. The testicular volume obtained from 3D MR imaging was approximately 60 mm3 in the control mice. A previous study showed the 3D reconstitution of serial testicular sections (50.4~63.2 mm3: average 55.8 mm3) in male mice of the C57BL/6 strain (Nakata et al., 2020). Owing to this result, we can confirm the validity of the non-invasive MR imaging in the present study. The T1-weighted 3D gradient-echo sequence supported our observations on the gross pathology of the testes: the total testicular volume in busulfan-treated mice was significantly smaller than in control mice. The seminiferous tubule volume matches the testicular volume because the ratio of the total testicular volume to the total seminiferous tubule volume is similar between individuals irrespective of testes size (Nakata et al., 2015). Histopathological analysis revealed that the significant reduction in testes weight among busulfan-treated mice was mostly due to spermatogenic cell depletion in the seminiferous tubules. On the other hand, imaging provides 3D quantitative data on tissue changes that cannot be easily acquired using traditional pathological approaches. In vivo 3D imaging of the testes can help predict the effects of pharmaceuticals, such as anti-cancer drugs, on testicular histopathology, even without dissection to contribute to a reduction in the number of animals used. In the future, MRI systems can provide useful temporal information to evaluate testicular toxicity in the same animals, potentially reducing the number of animals used in toxicological studies.
Finally, compared to that of control mice, the signal on T1-weighted images significantly increased in the testes of busulfan-treated mice. Several clinical studies have shown that high T1 signals (T1 shortening) are detected in high concentrations of macromolecules, such as in mucin-containing tumors (Gaeta et al., 2002). The oligosaccharides attached to the peptide chains within the thick mucin cause T1 shortening either by themselves or through water–macromolecule interactions, which can increase T1 signal intensity within tumors (Wei et al., 2022). Histopathological analysis revealed PAS-positive effusions in the interstitium of the testes of busulfan-treated mice. Given the principle of PAS staining, these effusions may be composed of glycoprotein-like substances. Therefore, the results of the staining may be related to T1 shortening due to a high concentration of glycoproteinaceous content.
In conclusion, the present study demonstrated that small animal imaging in vivo can detect morphological and anatomical changes in the testes, using an infertility mouse model to better understand non-clinical testicular toxicity. However, the study had limitations. The data obtained were insufficient for a time-course evaluation. Spermatogenesis is a continuous, cyclical, and synchronized process that occurs in the epithelium of the seminiferous tubules of the testes, spanning approximately 35 days in mice and 74 days in humans (Fayomi and Orwig, 2018). Chemicals may influence the testes at any point in the cycle. Therefore, it is difficult to predict when and where testicular toxicity may occur. Testicular toxicity should not be evaluated at only one time point but at many time points. Further investigations are needed to evaluate sequential non-invasive 3D imaging by MRI analysis. This will enable researchers to examine the longitudinal progression of the signs of testicular toxicity and its reversibility in the same animals treated with chemicals.
The authors thank Mr. Yoshiharu Tsuru, Mr. Itaru Higuchi, and Mr. Takahiro Aoki (Primetech Co. Ltd., Tokyo, Japan) for their technical assistance. We thank Editage (https://www.editage.jp) for their English language editing service. This study was supported by the Japan Agency for Medical Research and Development (grant number 21mk0101210j0001 to Satoshi Yokota).
Conflict of interestThe authors declare that there is no conflict of interest.
